Extended Data Figure 3: Genetic modulation of AMD1 in prostate cancer cells.

a, Technical setup of anti-AMD1 antibody using the indicated constructs or shRNAs in DU145 cells. AMD1^S229A mutant lacks processing ability and is expressed exclusively as a pro-enzyme (representative western blot out of three independent experiments). b, Schematic representation of tagged ectopic AMD1 processing. c, Impact of ectopic Myc-AMD1–HA expression on foci number in DU145 cells in vitro (n = 3 independent experiments). d–f, Impact of ectopic Myc-AMD1–HA expression on tumour volume (d, n = 8 tumours per condition), AMD1 protein levels (e, n = 3 tumours per condition), and dcSAM abundance (f, n = 5 tumours per condition) in DU145 xenografts grown for 43 days. Data in d are represented as box-and-whisker plot. f, Dot plot with the median and the interquartile range. g–o, Effect of constitutive silencing of AMD1 (g, i, mRNA levels; h–j, protein expression) on cell number (k, n), anchorage-independent growth (l, o), and dcSAM abundance (m) with two different hairpins in DU145 (g, h, k, l, m) and PC3 (i, j, n, o) cells (n = 3 or 4 independent experiments as indicated by dots). shSC, scramble short hairpin; sh3 and sh4, two different short-hairpins targeting AMD1. Mean ± s.e.m. p, Effect of doxycycline-inducible (100 ng ml⁻¹) AMD1 silencing on cell number in DU145 cells (sh3, n = 4; sh4 and sh5, n = 3 independent experiments as indicated by dots). q–s, Impact of AMD1-inducible silencing in tumour volume from DU145 xenografts (tumours analysed: sh3 no doxycycline, n = 12; sh3 doxycycline, n = 14; sh4 no doxycycline, n = 10; sh4 doxycycline, n = 7; sh5 no doxycycline, n = 10; sh5 doxycycline, n = 11). Box-and-whisker plot. t, Impact of AMD1-inducible silencing in dcSAM abundance in DU145 xenografts from q to s (n = 5 tumours). Median ± interquartile range. u, v, Dose-dependent effect of doxycycline on cell number in DU145 (u) and PC3 (v) cells (cell number measured at day 6) (n = 3 independent experiments as indicated by dots). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars, mean ± s.e.m. Red asterisk in western blots indicates non-specific band. Dashed lines indicate cell numbers in scramble short-hairpin-transduced cells. One-tailed t-test (c, g, i, k–p, u, v), and one-tailed Mann–Whitney U-test (d, f, q–t).